RESUMEN
Acidic ribosomal P-proteins form, in all eukaryotic cells, a lateral protuberance, the so-called 'stalk', which is directly involved in translational activity of the ribosomes. In Saccharomyces cerevisiae cells, there are four distinct P-proteins: P1A, P1B, P2A and P2B. In spite of the high level of their structural homology, they are not completely equivalent and may perform different functions. As yet, the protein-protein interactions between yeast P-proteins have not been fully defined. In this paper, the interplay between yeast P-proteins has been investigated by means of a two-hybrid system, chemical cross-linking and gel filtration. The data presented herein show that all P-proteins are able to form homo-oligomeric complexes. By analyzing hetero-interactions, we were able to detect strong interactions between P1A and P2B proteins. Additionally, the pair of P1B and P2A proteins is also able to form a hetero-complex, though at a very low efficiency. All P-proteins are phosphorylated by numerous protein kinases. Using the multifunctional protein kinase CK II, we have shown that incorporation of phosphate into P1A protein can exert its effect on the hetero-oligomerization process, namely by preventing the formation of the hetero-oligomer P1A-P/P2B. These findings are the first to show differences in the oligomerization behavior of the yeast P-proteins; moreover, they emphasize a significant impact of the phosphorylation on the formations of P-protein complex.
Asunto(s)
Proteínas Protozoarias , Proteínas Ribosómicas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Cromatografía en Gel , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Fosforilación , PlásmidosRESUMEN
The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The opioid peptides are widely distributed throughout the body, and they are generated during stress and inflammatory reaction. Opioids are involved in the communication between the immune and neuroendocrine systems. In the present study we have investigated the ability of both met-enkephalin and beta-endorphin to stimulate and prime the human neutrophils for enhanced chemiluminescence (CL) and chemotaxis induced with fMLP, OZ or PMA. We have also tested the effect of beta-endorphin and met-enkephalin on CD11a, CD11b, CD18 and CD16 molecule expression on PMN in vitro. PMN from ten healthy donors were incubated in vitro with different concentrations of beta-endorphin or met-enkephalin, and the CL response was evaluated with luminometer. To assess the effect of opioid peptides on CD11a, CD11b, CD18 and CD16 molecule expression the whole blood samples were incubated with different concentrations of the opioids, then the white cells were labelled with respective PE-conjugated MoAb and evaluated by flow cytometry. We have shown that: (1) met-enkephalin and beta-endorphin at physiological concentrations relevant to that of in vivo (10(-8) and 10(-6) M) enhanced fMLP, PMA or OZ stimulated chemiluminescence and induced chemotactic response, (2) High concentrations of beta-endorphin (10(-3) M) or met-enkephalin (10(-5) M) decreased the CL response of PMN in vitro, (3) The opioid peptides at lower concentrations resulted in CD11b and CD18 molecule up-regulation on neutrophils. We may conclude that opioid peptides in physiological concentration are involved in neutrophil priming whereas in higher concentration exert immunosuppressive potency. Opioid peptides like inflammatory cytokines may prime the neutrophils inflammatory response.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Encefalina Metionina/farmacología , Antígeno de Macrófago-1/biosíntesis , Neutrófilos/efectos de los fármacos , betaendorfina/farmacología , Animales , Antígenos CD18/biosíntesis , Células Cultivadas , Citometría de Flujo , Humanos , Integrina alfaXbeta2/biosíntesis , Mediciones Luminiscentes , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Ratones , Neutrófilos/metabolismo , Neutrófilos/fisiología , Receptores de IgG/biosíntesisRESUMEN
The 60S ribosomal subunits from Saccharomyces cerevisiae contain a set of four acidic proteins named YP1alpha, YP1beta, YP2alpha, and YP2beta. The genes for each were PCR amplified from a yeast cDNA library, sequenced, and expressed in Escherichia coli cells using two expression systems. The first system, pLM1, was used for YP1beta, YP2alpha, and YP2beta. The second one, pT7-7, was used for YP1alpha. Expression in both cases was under the control of a strong inducible T7 promoter. The amount of induced recombinant proteins in the host cells was around 10 to 20% of the total soluble bacterial proteins. A new protocol for purification of all four recombinant proteins was established. The preliminary steps of purification were done by ammonium sulfate precipitation (YP1alpha, YP1beta) or NH4Cl/ethanol extraction (YP2alpha, YP2beta). The recombinant proteins were then purified to apparent homogeneity by only two steps of classical chromatographies, ion exchange (DEAE-cellulose) and gel filtration (Sephacryl S-200). Isoelectrofocusing analysis of YP2alpha and YP2beta showed the pIs of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pI of YP1alpha is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1beta was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1beta'. This was proved by isoelectrofocusing gel analysis.
Asunto(s)
Clonación Molecular/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Saccharomyces cerevisiae/genética , Sulfato de Amonio , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Indicadores y Reactivos , Focalización Isoeléctrica , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Ribosómicas/biosíntesis , Saccharomyces cerevisiae/metabolismoRESUMEN
The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function. The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast. Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator. This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1. Additionally, truncated proteins of YP1alpha and YP1beta were analyzed. The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not. Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reporteros , Operón Lac , Fosfoproteínas/química , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Activación TranscripcionalRESUMEN
BACKGROUND: Topical application of growth factors to wounds has proven to be suboptimal in achieving epithelial growth and accelerating healing. We propose transfection of fibroblasts with a gene for acidic fibroblast growth factor (aFGF) which will allow continuous, local delivery of the growth factor to wounds, ulcerative lesions, or healing tissues. METHODS: We utilized a pMEXneo vector containing the human aFGF gene with a secretory signal sequence from the hst/KS3 gene to obtain continuous secretion of therapeutic doses of aFGF. NIH 3T3 fibroblasts were transfected using a liposomal transfection reagent and grown in selective media. RESULTS: Dot blot hybridization with labeled complementary DNA probes revealed the presence of plasmid DNA in transfected but not wild type fibroblasts. Intracellular concentrations of aFGF remained low in transfected cells; however, the media contained high levels (32 +/- 7 nM) of aFGF as measured by ELISA. Concentrations of aFGF capable of stimulating cell proliferation were maintained for several weeks. CONCLUSIONS: The aFGF cDNA was transcribed and translated into a functional polypeptide that is secreted from NIH 3T3 cells at physiologically significant concentrations. Stable transfection with a eukaryotic vector which induces secretion of aFGF at levels promoting cell growth holds promise for clinical application in wounds or healing tissue. Transfection could be achieved by topical or endoscopic injection of this type of vector.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/genética , Técnicas de Transferencia de Gen , Plásmidos , Cicatrización de Heridas/fisiología , Células 3T3/citología , Células 3T3/metabolismo , Animales , ADN Complementario , Expresión Génica , Immunoblotting , Ratones , Transfección/métodosRESUMEN
The current study examines the stimulation of healing processes and signal transduction that is mediated by insulin-like growth factor-I (IGF-I) in an ex vivo esophageal explant model when using tyrphostin inhibition of receptor tyrosine kinase. The explant model provides a 3-dimensional cellular environment of multiple interacting cells isolated from the neural and vascular supply. Tyrphostins previously characterized for their interactions with epithelial growth factor (EGF) receptor-associated protein tyrosine kinases were tested for their potential effects on IGF-I growth-promoting activity. Explants of rabbit esophagus were incubated in media with or without IGF-I. Tyrphostins 1, 23, 25, 46, 47, 51, and 63 were added. We assessed DNA synthesis by tritiated thymidine incorporation. Outgrowth from the edge of the primary mucosa of the explant was evaluated on histologic sections, and cell proliferation was confirmed with immunohistology. IGF-I increased the incorporation of tritiated thymidine by 50% to 100%. Tyrphostins 23 and 47 eliminated IGF-I-induced proliferation in a dose-dependent manner. Tyrphostins 25, 46, and 51--along with negative controls tyrphostin 1 and tyrphostin 63--were ineffective, inasmuch as IGF-I-stimulated growth remained unchanged in their presence. Proliferative activity demonstrated by PCNA staining was confined to new mucosa. Two of 5 tyrphostins originally developed as EGF receptor protein tyrosine kinase inhibitors were effective in inhibiting the actions of exogenous IGF-I. We conclude that IGF-I stimulation may play an important role in repair processes in the esophagus and that this stimulation can be inhibited by using specific tyrphostins.
Asunto(s)
Enfermedades del Esófago/patología , Esófago/patología , Factor I del Crecimiento Similar a la Insulina/farmacología , Tirfostinos , Animales , Catecoles/farmacología , División Celular , Técnicas de Cultivo , ADN/biosíntesis , Esófago/química , Inhibidores de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Membrana Mucosa/patología , Nitrilos/farmacología , Antígeno Nuclear de Célula en Proliferación/análisis , Conejos , Regeneración , Transducción de SeñalRESUMEN
A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.
Asunto(s)
Dioxigenasas , Neurospora crassa/enzimología , Oxigenasas/aislamiento & purificación , Acetona/metabolismo , Radicales Libres , Cinética , Peso Molecular , Oxigenasas/metabolismo , Especificidad por SustratoRESUMEN
Proliferation of esophageal mucosal cells is important in repair of reflux-induced injury. We studied the effects of EGF, IGF-I, and IGF-I/binding protein-3 (BP-3) complex on immortalized esophageal epithelial (HET-1A) cells and searched for synergy between the growth factors. HET-1A cells were plated at 5 x 10(4) in 12 well plates. After 2 days in optimal media, they were maintained in basal media with or without the test peptides: EGF at 0.05-5 nM, IGF-I at 0.1-10 nM, IGF-I/BP-3 at 0.1-10 nM, and a combination of EGF at 5 nM and IGF-I or IGF-I/BP-3 at 1 and 10 nM. In comparison to basal media EGF and IGF-I stimulated cell proliferation over baseline at 5 and 10 nM, respectively. The combination of EGF at 5 nM and IGF-I at 1 and 10 nM worked synergistically, increasing cell counts over baseline to 10.3 +/- 0.2 and 14.6 +/- 0.8 x 10(5), respectively. The calculated additive effect of EGF and IGF-I at 1 and 10 nM individually increased cell counts to 8.2 +/- 0.2 and 10.4 +/- 0.6 x 10(5), respectively. The difference between the observed and the calculated values was significant at P < 0.05, ANOVA, Turkey test. IGF-I/BP-3 complex enhanced this synergy at low levels of IGF-I but not at 10 nM IGF-I. EGF, IGF, and IGF-I/BP-3 independently promote HET-1A proliferation. IGF and EGF in combination demonstrate synergism with potentiated interaction presumably because of their different roles in the cell cycle, EGF being a competence factor and IGF being a progression factor. This combination may have potential as a treatment for esophageal mucosal injury, and IGF-I/BP-3 may further enhance their benefit.
Asunto(s)
Factor de Crecimiento Epidérmico/administración & dosificación , Esófago/citología , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Línea Celular , Sinergismo Farmacológico , Células Epiteliales , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismoRESUMEN
We have isolated the gene encoding 2-nitropropane dioxygenase from Hansenula mrakii, an FAD enzyme that catalyzes the oxygenative denitrification of various anionic nitroalkanes. The gene contained an open reading frame consisting of 1122 nucleotides corresponding to 374 amino acid residues. The protein molecular mass was estimated to be 41,466 Da, which was similar to the subunit molecular mass of the enzyme determined by SDS/PAGE. Several FAD enzymes such as D-amino acid oxidase and glucose oxidase also catalyze the oxidation of nitroalkanes as a side-reaction, although not so efficiently [Kido, T. & Soda, K. (1984) Arch. Biochem. Biophys. 234, 468-475]. However, we found no proteins in the databases (GenBank, EMBL, PIR and SWISS-PROT) which are homologous to 2-nitropropane dioxygenase of H. mrakii in primary structure. No protein motifs, including a nucleotide-binding motif, GXGXXG, were found in PROSITE, a database of biologically significant protein sites and patterns. Accordingly, 2-nitropropane dioxygenase is a new type of flavoprotein with a unique structure.
Asunto(s)
Dioxigenasas , Oxigenasas/química , Pichia/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Enzimas de Restricción del ADN , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Datos de Secuencia Molecular , Oxigenasas/genética , Pichia/genética , Biosíntesis de Proteínas , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
Two yeast casein kinase type-1 species of 45 kDa and 27 kDa (CK1) were purified to apparent homogeneity and used for investigation of their immunological affinity. Antisera against the two kinases were isolated; the antibody against the 45 kDa kinase did not react with the 27 kDa enzyme. The 27 kDa casein kinase was recognized only by its own antibody. The obtained data strongly suggest that the low molecular mass CK-1 is not a proteolytic product of the 45 kDa kinase species.